Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue

The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1 : contributions of the pore domains

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb+ or Na+ replaced extracellular K+. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels nonselective. We interpret the effects of these—and of other mutations—in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter

Union effectiveness: In Search of the Holy Grail

YesThis article revisits the concept of union effectiveness and proposes a conceptual model to inform its study and application. Previous conceptual and empirical work is examined to identify key strengths and weaknesses, and to relate the union effectiveness concept to union renewal and other key concepts. This leads to the proposal of a Goal-System framework that builds and improves on prior research

Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage

<p>Abstract</p> <p>Background</p> <p>Blood specimen collection at an early study visit is often included in observational studies or clinical trials for analysis of secondary outcome biomarkers. A common protocol is to store buffy coat specimens for future DNA isolation and these may remain in frozen storage for many years. It is uncertain if the DNA remains suitable for modern genome wide association (GWA) genotyping.</p> <p>Methods</p> <p>We isolated DNA from 120 Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial buffy coats sampling a range of storage times up to 9 years and other factors that could influence DNA yield. We performed TaqMan SNP and GWA genotyping to test whether the DNA retained integrity for high quality genetic analysis.</p> <p>Results</p> <p>We tested two QIAGEN automated protocols for DNA isolation, preferring the Compromised Blood Protocol despite similar yields. We isolated DNA from all 120 specimens (yield range 1.1-312 ug per 8.5 ml ACD tube of whole blood) with only 3/120 samples yielding < 10 ug DNA. Age of participant at blood draw was negatively associated with yield (mean change -2.1 ug/year). DNA quality was very good based on gel electrophoresis QC, TaqMan genotyping of 6 SNPs (genotyping no-call rate 1.1% in 702 genotypes), and excellent quality GWA genotyping data (maximum per sample genotype missing rate 0.64%).</p> <p>Conclusions</p> <p>When collected as a long term clinical trial or biobank specimen for DNA, buffy coats can be stored for up to 9 years in a -80degC frozen state and still produce high yields of DNA suitable for GWA analysis and other genetic testing.</p> <p>Trial Registration</p> <p>The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial is registered with ClinicalTrials.gov, number <a href="http://www.clinicaltrials.gov/ct2/show/NCT00000620">NCT00000620</a>.</p

Lubiprostone Stimulates Duodenal Bicarbonate Secretion in Rats

Lubiprostone, a bicyclic fatty acid, is used for the treatment of chronic constipation. No published study has addressed the effect of lubiprostone on intestinal ion secretion in vivo. The aim of this study was to test the hypothesis that lubiprostone augments duodenal HCO3 − secretion (DBS). Rat proximal duodenal loops were perfused with pH 7.0 Krebs, control vehicle (medium-chain triglycerides), or lubiprostone (0.1–10 μM). We measured DBS with flow-through pH and CO2 electrodes, perfusate [Cl−] with a Cl− electrode, and water flux using a non-absorbable ferrocyanide marker. Some rats were pretreated with a potent, selective CFTR antagonist, CFTRinh-172 (1 mg/kg, ip), 1 h before experiments. Perfusion of lubiprostone concentration dependently increased DBS, whereas net Cl− output and net water output were only increased at 0.1 μM, compared with vehicle. CFTRinh-172 reduced lubiprostone (10 μM)-induced DBS increase, whereas net Cl− output was also unchanged. Nevertheless, CFTRinh-172 reduced basal net water output, which was reversed by lubiprostone. Furthermore, lubiprostone-induced DBS was inhibited by EP4 receptor antagonist, not by an EP1/2 receptor antagonist or by indomethacin pretreatment. In this first study of the effect of lubiprostone on intestinal ion secretion in vivo, lubiprostone stimulated CFTR-dependent DBS without changing net Cl− secretion. This effect supports the hypothesis that Cl− secreted by CFTR is recycled across the apical membrane by anion exchangers. Recovery of water output during CFTR inhibition suggests that lubiprostone may improve the intestinal phenotype in CF patients. Furthermore, increased DBS suggests that lubiprostone may protect the duodenum from acid-induced injury via EP4 receptor activation

Beyond climate envelopes: effects of weather on regional population trends in butterflies

Although the effects of climate change on biodiversity are increasingly evident by the shifts in species ranges across taxonomical groups, the underlying mechanisms affecting individual species are still poorly understood. The power of climate envelopes to predict future ranges has been seriously questioned in recent studies. Amongst others, an improved understanding of the effects of current weather on population trends is required. We analysed the relation between butterfly abundance and the weather experienced during the life cycle for successive years using data collected within the framework of the Dutch Butterfly Monitoring Scheme for 40 species over a 15-year period and corresponding climate data. Both average and extreme temperature and precipitation events were identified, and multiple regression was applied to explain annual changes in population indices. Significant weather effects were obtained for 39 species, with the most frequent effects associated with temperature. However, positive density-dependence suggested climatic independent trends in at least 12 species. Validation of the short-term predictions revealed a good potential for climate-based predictions of population trends in 20 species. Nevertheless, data from the warm and dry year of 2003 indicate that negative effects of climatic extremes are generally underestimated for habitat specialists in drought-susceptible habitats, whereas generalists remain unaffected. Further climatic warming is expected to influence the trends of 13 species, leading to an improvement for nine species, but a continued decline in the majority of species. Expectations from climate envelope models overestimate the positive effects of climate change in northwestern Europe. Our results underline the challenge to include population trends in predicting range shifts in response to climate change

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

<p>Abstract</p> <p>Background</p> <p>In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG) and induces Ca²⁺release from endoplasmic reticulum (ER) stores.</p> <p>Methods</p> <p>In the present study, we monitored the cytosolic Ca²⁺transients using the UV light photolysis technique to uncage caged Ca²⁺or caged IP₃into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca²⁺receptors present in the ER, and measured their Ca²⁺dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).</p> <p>Results</p> <p>We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP₃R) dependent-Ca²⁺response following both correcting treatments compared to uncorrected cells in such a way that Ca²⁺responses (CF+treatment <it>vs </it>wild-type cells) were normalized. This normalization of the Ca²⁺rate does not affect the activity of Ca²⁺-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP₃R1, we observed a decrease of the implication of IP₃R1 in the Ca²⁺response in CF corrected cells. We observed a similar Ca²⁺mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP₃R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.</p> <p>Conclusion</p> <p>These results suggest reversal of the IP₃R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP₃R Ca²⁺release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.</p

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Improved annotation with <i>de novo</i> transcriptome assembly in four social amoeba species

Background: Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA sequencing (RNA-seq) data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species. Results: An initial de novo transcriptome assembly was generated by Trinity for each species and then refined with Program to Assemble Spliced Alignments (PASA). The completeness and quality were assessed with the Benchmarking Universal Single-Copy Orthologs (BUSCO) and Transrate tools at each stage of the assemblies. The final datasets of 11,315-12,849 transcripts contained 5,610-7,712 updates and corrections to >50% of existing gene models including changes to hundreds or thousands of protein products. Putative novel genes are also identified and alternative splice isoforms were observed for the first time in P. pallidum, D. lacteum and D. fasciculatum. Conclusions: In taking a whole transcriptome approach to genome annotation with empirical data we have been able to enrich the annotations of four existing genome sequencing projects. In doing so we have identified updates to the majority of the gene annotations across all four species under study and found putative novel genes and transcripts which could be worthy for follow-up. The new transcriptome data we present here will be a valuable resource for genome curators in the Dictyostelia and we propose this effective methodology for use in other genome annotation projects

Comparative transcriptome sequencing of germline and somatic tissues of the Ascaris suum gonad

<p>Abstract</p> <p>Background</p> <p><it>Ascaris suum </it>(large roundworm of pigs) is a parasitic nematode that causes substantial losses to the meat industry. This nematode is suitable for biochemical studies because, unlike <it>C. elegans</it>, homogeneous tissue samples can be obtained by dissection. It has large sperm, produced in great numbers that permit biochemical studies of sperm motility. Widespread study of <it>A. suum </it>would be facilitated by more comprehensive genome resources and, to this end, we have produced a gonad transcriptome of <it>A. suum</it>.</p> <p>Results</p> <p>Two 454 pyrosequencing runs generated 572,982 and 588,651 reads for germline (TES) and somatic (VAS) tissues of the <it>A. suum </it>gonad, respectively. 86% of the high-quality (HQ) reads were assembled into 9,955 contigs and 69,791 HQ reads remained as singletons. 2.4 million bp of unique sequences were obtained with a coverage that reached 16.1-fold. 4,877 contigs and 14,339 singletons were annotated according to the <it>C. elegans </it>protein and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. Comparison of TES and VAS transcriptomes demonstrated that genes participating in DNA replication, RNA transcription and ubiquitin-proteasome pathways are expressed at significantly higher levels in TES tissues than in VAS tissues. Comparison of the <it>A. suum </it>TES transcriptome with the <it>C. elegans </it>microarray dataset identified 165 <it>A. suum </it>germline-enriched genes (83% are spermatogenesis-enriched). Many of these genes encode serine/threonine kinases and phosphatases (KPs) as well as tyrosine KPs. Immunoblot analysis further suggested a critical role of phosphorylation in both testis development and spermatogenesis. A total of 2,681 <it>A. suum </it>genes were identified to have associated RNAi phenotypes in <it>C. elegans</it>, the majority of which display embryonic lethality, slow growth, larval arrest or sterility.</p> <p>Conclusions</p> <p>Using deep sequencing technology, this study has produced a gonad transcriptome of <it>A. suum</it>. By comparison with <it>C. elegans </it>datasets, we identified sets of genes associated with spermatogenesis and gonad development in <it>A. suum</it>. The newly identified genes encoding KPs may help determine signaling pathways that operate during spermatogenesis. A large portion of <it>A. suum </it>gonadal genes have related RNAi phenotypes in <it>C. elegans </it>and, thus, might be RNAi targets for parasite control.</p